Application Details

Application Details

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Sample Volume

100 μL

Assay Time

4.5 h

Plate

Pre-coated

Protocol

The Mouse VEGF-A ELISA kit is a solid phase sandwich ELISA (enzyme-linked immunosorbent assay) for the quantitative measurement of VEGF-A in mouse serum and plasma. An antibody specific for mouse VEGF-A was coated on a 96-well plate. Standards and samples are added to the wells and any VEGF-A present binds to the immobilized antibody. The wells are washed and biotinylated anti-mouse VEGF-A antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is added to the wells. The wells are again washed and TMB substrate solution is added, which produces a blue color in direct proportion to the amount of VEGF-A present in the initial sample. The Stop Solution changes the color from blue to yellow, and the microwell absorbances are read at 450 nm

Streptavidin-HRP: Briefly spin the HRP-Streptavidin concentrate vial and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 160-fold with 1X Assay Diluent.

Sample: Levels of the target protein may vary among different specimens. Optimal dilution factors for each sample must be determined by the investigator, the recommended dilution for serum and plasma is 1: 2.

Sample Collection

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 4 °C before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or citrate or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 x g at 2-8 °C within 30 minutes of collection. Assay immediately or aliquot and store samples at -20 °C or -80 °C. Avoid repeated freeze-thaw cycles. Cell Culture Supernatant: Centrifuge samples for 20 minutes at 1000 x g. Remove particulates and assay immediately or store samples in aliquot at -20 °C or -80 °C for later use. Avoid repeated freeze/thaw cycles.

Assay Procedure

All reagents must be brought to room temperature (18-25 °C) prior to use

Prepare all reagents, detection anytibody, standard and samples as directed in the respective sections.

Remove required quantity of test strips/wells, place in well holder.4. Add 100 μL of each standard and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking.

Decant or aspirate contents of wells. Wash wells by filling with at least 300 μL/well prepared wash buffer followed by decanting/aspirating. Repeat wash 4 times for a total of 5 washes. After the last wash, blot plate on absorbent paper to remove residual buffer.

Add 100 μL of 1X prepared biotinylated antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.7. Discard the solution. Repeat the wash as in step

10. Add 100 μL of TMB One-Step Substrate Reagent to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.11. Add 50 μL of Stop Solution to each well. Read absorbance at 450nm within 30 minutes of stopping reaction. If wavelength correction is available, subtract the optical density readings at 570nm from readings at 450nm.

Calculation of Results

Average the duplicate readings for each standard and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the target antigen concentration versus the log of the O.D. and the best fit line can be determined by regression analysis. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.